Vibrio vulnificus is an opportunistic human pathogen capable of causing fatal primary septicemias or necrotizing wound infections. Septicemia occurs in patients that are immunocompromised or suffering from hemochromatosis or with other underlying liver disorders such as cirrhosis and alcoholic liver disease. The common theme in most of these patients is that iron is present at higher than physiological level. We believe that gene regulation of V. vulnificus CMCP6 depends on many factors according to the changes in environmental conditions i.e. high iron, nutrients concentration, and oxygen availability during the process of infection. In this application we propose to dissect the specific mechanisms that govern their expression in V. vulnificus, in vitro and in vivo. The specific aims to achieve these goals are: 1) In vitro regulation of genes whose expression is affected by the iron concentration of the medium and/or components of human serum of compromised patients. In this aim we identify factors in addition to the high iron concentration of the serum play a role in the expression of virulence genes expressed in patient sera. 2) Analysis of HlyU, a global transcription regulator and virulence factor of V. vulnificus expressed at both high and iron limiting conditions. We demonstrate that HlyU in addition to being an important virulence factor is also a global transcriptional regulator that at both iron-rich and iron limiting conditions controls the expression of many virulence-genes, some of them highly expressed only at high iron conditions. In this aim we propose to use a combination of transcriptional and translational fusions as well as gel shift and DNAsel protection experiments with the purified HlyU protein. 3) Analysis of the in vivo expression of V. vulnificus genes in two different mouse models. In this specific aim we propose to use recombination-based in vivo technology (RIVET) to identify V. vulnificus genes that are expressed specifically in vivo during infection of the iron-overloaded mouse model as compared to those induced after infecting the normal mouse. The genes identified using both mouse models as well as those identified in vitro will be characterized by mutagenesis and virulence experiments. Thus, we expect to obtain a comprehensive picture of the physiology of this bacterium during in vivo as compared to in vitro growth. These studies will lead to an enhanced understanding of the pathogenesis of V. vulnificus infections in particular and of bacterial virulence in general.